Primer sequences used in this work.

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.</div

RNASeq of infected HFFs.

Differential gene analysis and interaction analysis of human-aligned transcripts. IFNγ treatment is denoted as yes or no. For the interaction analysis, sheet names represent the IFNγ effect for the first strain relative to the IFNγ effect for the second strain (i.e., WT|IFN_vs_dGRA57|IFN represents +IFNγ vs -IFNγ in the RHΔKu80 group relative to +IFNγ vs -IFNγ in the RHΔGRA57 group). (XLSX)</p

Opera Phenix image acquisition parameters and Harmony analysis sequence used for automated ubiquitin recruitment analysis.

Opera Phenix image acquisition parameters and Harmony analysis sequence used for automated ubiquitin recruitment analysis.</p

Antibodies used for immunofluoresence assays.

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.</div

Inhibition of parasite egress in the first 3 h of infection does not restore ubiquitination or survival of ΔGRA57 vacuoles.

(A) Recruitment of total ubiquitin to Toxoplasma vacuoles at 3 h postinfection, with the addition of 5 μm Compound 2 at 30 min postinfection to inhibit parasite egress. HFFs were pre-stimulated with 100 U/ml IFNγ for 24 h, infected with indicated lines for 30 min prior to addition of Compound 2. HFFs were fixed at 3 h postinfection and stained for total ubiquitin. Recruitment was automatically counted using high-content imaging and analysis. (B) Parasite survival in IFNγ-stimulated HFFs at 3 h postinfection, with the addition of 5 μm Compound 2 at 30 min postinfection to inhibit parasite egress. Parasite numbers were quantified through automated high-content imaging, with survival calculated as the percentage of intracellular parasites in IFNγ-stimulated cells relative to the total in unstimulated cells. p-Values were calculated by paired two-sided t test. *, p p p p S10 Data. (TIF)</p

Raw data for Cytation plate reader IFNγ survival assays.

(A) HFFs, (B) MEFs, and (C) HFFs +/− tryptophan supplementation. Total mCherry area +/− IFNγ per biological replicate. (XLSX)</p

Raw data for Compound 2 egress inhibition assays.

(A) Percentage of vacuoles with ubiquitin recruitment +/− IFNγ and +/− Compound 2 per biological replicate. (B) Survival percentages at 3 h postinfection calculated from number of intracellular Toxoplasma per biological replicate. (XLSX)</p

GRA57 deletion does not affect formation of the IVN.

HFFs monolayers were infected with the indicated strains for 24 h prior to fixation and preparation for transmission electron microscopy (TEM). (TIF)</p

GRA57 is an intravacuolar protein that contributes to parasite survival of IFNγ in HFFs.

(A) IFA of RHΔGRA57 and RHΔGRA57::GRA57-HA lines generated for this study. HA-tagged GRA57 co-localises with GRA2—a marker of the IVN. Scale bar = 3 μm. (B) Western blot analysis of GRA57-HA shows it is a 250 kDa protein that is relatively highly expressed. (C and D) Live restriction assays in (C) HFFs and (D) MEFs using mCherry fluorescence area as a readout for parasite survival. Host cells were pre-stimulated for 24 h with 100 U/ml IFNγ or left untreated then infected in technical triplicate with the indicated parasite strains for 48 h (HFFs) or 24 h (MEFs) at an MOI of 0.3. Infected cells were then imaged live on a Cytation plate reader. Total mCherry signal area per well was measured to determine parasite growth in IFNγ-stimulated relative to unstimulated cells. Data displayed as median survival with individual biological replicates overlayed. p-Values were calculated by paired two-sided t test. **, p S3 Data. HFF, human foreskin fibroblast; MEF, mouse embryonic fibroblast.</p

GRA57-HA partially co-localises with the PVM marker GRA3.

GRA57-HA partially co-localises with the PVM marker GRA3.</p